Sulfonated sclerostin, antibodies, epitopes and methods for identification and use therefor

ABSTRACT

Provided are antibodies that bind to: a sulfonated epitope of the protein Sclerostin, to Sclerostin portions comprising a sulfonated amino acid and to dimerized forms of Sclerostin. Further provided are compositions and peptides comprising a sulfonated epitope of sclerostin. Also provided by this invention are methods for production of such antibodies, both active and passive, and methods for identifying antibodies specific for sulfonation sites in Sclerostin and other antibodies which discriminate between sulfonated and unsulfonated forms of sclerostin. Physical and virtual screening processes are provided in this invention for identifying compounds which disrupt or inhibit sulfonation and the interaction between Sclerostin and binding partners. The antibodies and compositions of the present invention are useful in diagnostic and therapeutic applications directed to Sclerostin-related disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/802,447, filed Jun. 7, 2010, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to the field of immunotherapy, the discovery andapplication of pharmacological agents, and to the diagnosis andtreatment of Sclerostin-related disorders.

All patents, patent applications, patent publications, scientificarticles and the like, cited or identified in this application, arehereby incorporated by reference in their entirety in order to describemore fully the state of the art to which the present invention pertains.

BACKGROUND OF THE INVENTION

Although the nature of a protein is dictated primarily by the particularamino acid sequences derived from transcription of its nucleic acidcoding sequence, there are post-transcriptional processes that may alsoaffect its properties. Some of these modifications are large scalerearrangements such as: (a) conversion of an inactive pro-enzyme into anactive form by removal of part of an amino acid sequence; (b) proteasedigestion of a composite protein into individual segments with variedfunctions as seen in some viral proteins (for instance, the polyproteinof HIV); or (c) removal of an internal amino acid sequence (an intein)by protein splicing. In addition to these cleavage processes,modification of individual amino acids can take place by enzymaticaddition of functional groups such as methyl, acetyl, phosphate,glycosyl, palmitoyl, sulfonate and ubiquitin groups.

The difference in functionality caused by these modifications can induceradical differences in properties. For instance, proinsulin is aninactive enzyme that is only found in its active form (insulin) afterproteolytic cleavage transforms the protein into separate peptide chainsconnected by disulfide bonds. In another instance, the addition of aubiquitin moiety doesn't necessarily affect its enzymatic functions butgenerates a signal for degradation of the “tagged” protein. Evenrelatively modest alterations, such as acetylation and phosphorylationof one or more amino acids in a protein, can induce remarkable changesin the properties of a protein target. The importance of both of theseprocesses in controlling levels of activities within cells by suchmodifications can be seen by the abundance of substrate specificversions of each of these family of proteins (acetylases and kinases)within a cell. Further control is exerted by the action of proteins thatreverse these changes, i.e., de-acetylases and phosphatases. Thesemodifications can result in an increase or a decrease in the activitylevel of the target protein and/or a change in its physical locale.

Although the kinase and acetylase modifications are well known areas ofresearch, the importance of sulfonation is receiving increasedattention. For recent reviews see Stone et al., 2009 New Biotechnology25; 299-317 and Monigatti et al., 2006 Biochim Biophys Acta 17641904-1913). Sulfonation of Tyrosines is believed to take place in about1% of the Tyrosines in proteins and appears to facilitateprotein-protein interactions (Baeuerle and Huttner 1985 JBC 260;6434-6439, Kehoe and Bertozzi 2000 Chem Biol 7; R57-R61). Of specialinterest the connection between sulfonation with receptors and theirligands, since the enzymes, TPST-1 and TPST-2, responsible forsulfonation are localized in the Golgi apparatus. Although these havebeen observed to be mostly cytokine receptors and their ligands, it hasbeen recently noted that unsulfonated Wnt does not generate as strong asignal as sulfonated Wnt, presumably due to a differential ability ofthe unsulfonated ligands to bind the LRP5/6 receptors that are involvedin the Wnt signaling system (Cha et al., 2009 Current Biol 19;1573-1580). In addition to Tyrosine, evidence has become available thatSerine and Threonine are also potential sites, although at the presenttime it is not known if this is carried out by the same enzymes (TPST-1and TPST-2) that modify Tyrosines or if some enzyme or enzymes areresponsible (Medzihradszky et al., 2004 Molec Cell Proteomics 3;429-440).

Testing for the presence of sulfonation modifications in a protein canbe carried out using various methods (for reviews, see Monigatti et al.2006, Stone et al. 2009 and Seibert and Sakmar 2007 Polymer 90;459-477). The two most popular methods for this type of analysis is theuse of mass spectrometry (MS), or antibodies that are specific forSulfo-Tyr. With regard, to mass spectrometry, definitive answers on thepresence of sulfonated Tyrosines can be achieved, but due to thelability of the bond between the sulfonate group and Tyrosine, specialmodifications have to be made to the standard mass spectrometryprotocols (Drake and Hortin, 2010 Int J Biochem Cell Biol 42; 174-179).In a more biological approach, antibodies have been developed that candetect the presence of sulfonated Tyrosine residues. Antibodies havebeen developed that can detect the presence of sulfonated Tyrosine'sregardless of the particular peptide sequence they are embedded within(Kehoe et al., 2006 Molec Cell Proteomics 5; 2350-2363; Hoffhines etal., 2006 J. Biol Chem 281; 37,877-37,887). The general nature of theirrecognition allows a wide variety of different proteins to be recognizedas long as they contain a sulfonated Tyrosine. In many cases, proteinshave to be isolated or separated for this type of analysis to observeindividual effects, since there is no discrimination between thedifferent sulfonated proteins by such antibodies. For instance, theextent of sulfonation can be determined for individual isolated proteinsof interest or patterns of a group of proteins can be analyzed. On theother hand, antibodies have been developed for a specific protein with asulfonated Tyrosine. These antibodies can detect differences betweensulfonated and non-sulfonated forms and can identify the presence of thesulfonated protein in a mixture of other proteins (Bundgaard et al.,2008 Methods Mol Bio 446; 47-66). The specificity of the epitoperequires that a new antibody has to be developed for each particularprotein of interest.

As information has accumulated concerning the amino acid sequences thatare used as substrates for sulfonation, it has become clear that thereis no simple consistent recognition sequence (see Niehrs et al., 1990JBC 265; 8525-8532, Bundgaard et al., 1997 JBC 272; 31,700-31,705 forinstance). A computer program called “Sulfinator” has been createdrecently that is capable of analyzing protein sequences and predictingthe presence or absence of sulfonation sites (Monigatti et al. 2002Bioinformatics 18; 769-770). The program achieves its highest accuracyonly when proteins are tested that are either receptors, or ligands forreceptors, because these are proteins that are processed through theGolgi apparatus where the TPST-1 and TPST-2 enzymes are localized.Proteins that are cytosolic in nature are physiologically irrelevantsince even if they have appropriate sequences they would never come intocontact with the Tyrosine sulfotransferases. The Sulfinator does notdetect the extent of sulfonation.

In detecting the extent of sulfonation, experiments have shown that evenproteins that are substrates for sulfonation do not always represent ahomogeneous population with complete sulfonation. For instance, gastrinpeptides which are easily sulfonated show a mixed population of bothsulfonated and unsulfonated forms in roughly equal proportions (Hilstedand Rehnfeld 1987 JBC 262; 16,953-16,957). In another instance, theremay be tissue specific differentiation on the extent of Tyrosinesulfonation of Chromogranin A that depends upon whether it is made inparathyroid or adrenal cells (Gorr and Cohn, 1999, JBC 265; 3012-3016).Different effects have also been observed for proteins such asgastrin/cholecystokinin peptides and their precursors where varyingdegrees of modification are seen during ontogenesis and pathogenesis ofcertain diseases (Rehfeld et al., 1989 Biochimie 70; 25-31).Furthermore, in certain circumstances, such as in the expression ofcloned recombinant proteins, there may be undersulfonation of proteinsthat would otherwise be completely modified (Seibert and Sakmar 2008Biopolymers 90; 459-477).

Although extensive efforts have been made in searching forpharmaceutical agents that affect kinase activity, compounds that affectsulfonation modifications have only recently attracted attention (forinstance, see Hemmerich et al., 2004 Drug Discovery Today 9; 967-975).The potential utility of influencing sulfonation reactions can be seen,however, by recent discoveries that CCR5, one of the receptors forrecognition of HIV, is sulfonated. The importance of this modificationcan be seen by results with chlorate (an inhibitor of Tyrosinesulfonation), where the presence of this factor decreases the affinityof gp120/CD4 complexes towards the CCR5 receptor (Farzan et al., 1999Cell 96; 667-676). Although there are instances where the presence of asulfonation modification may enhance binding, there are also numerousinstances where there is actually an absolute requirement forsulfonation to have taken place in order for certain proteins to havebiological activity (Farzan et al., 2001 J Exp Med 193; 1059-1065,Costaglia et al. 2002 EMBO J 21; 504-513, Gao et al., 2003 JBC 278;37902-37908, Gutierrez et al., 2004 JBC 279; 14726-14733, Hirata et al.,2004 JBC 279; 51775-51782, Fieger et al., 2005 FASEB J 19; 1926-1928 andColvin et al., 2006 Molec Cell Biol 26; 5838-5849).

Furthermore, in vitro studies also show the importance of sulfonationwith regard to binding of gp120/CD4 complexes with CCR5 peptides(Cormier et al., 2000 Proc. Nat. Acad. Sci USA 97; 5762-5767). As such,it has been recognized that the disruption of the sulfonation of CCR5may be a treatment for HIV infection and disease processes. In anotherexample, Liu et al. 2008 (Am J Resp Cell Molec Biol 38; 738-743)hypothesized that sulfonation was a general feature of cytokinereceptors and found that at least 10 different cytokine receptors thatare involved in asthma and chronic obstructive pulmonary disease (COPD)are sulfonated. On this basis, the authors concluded that incorporationof this discovery into the structural design of receptor antagonistsmight show value in the development of effective drug therapies forasthma, COPD and similar inflammatory lung diseases.

Changes in sulfonation patterns have also been found for tumour derivedenzymes (Itkonen et al., 2007 FEBS Journal 275; 289-301 and a dependencyon sulfonation has been shown for binding of P-selectin to cancer cells(Ma and Geng 2002 J Immunol 168; 1690-1696) and tumorigenesis (Feng etal., 2010 J Vir 84; 3351-3361).

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that Sclerostinpossesses post-translational modifications where at least two aminoacids are sulfonated in vivo. It is known that sulfonation modificationsare frequently involved in protein-protein interactions and as such, ithas been found that Sclerostin exhibits sulfonation-dependentalterations in a variety of biological properties that are likelyderived from protein-protein interactions. These include effects on thebinding of Sclerostin to the LRP5 receptor as well as differentialeffects upon Wnt signaling as judged by inhibition of blockage byvarious amounts of Sclerostin on the induction of alkaline phosphataseby Wnt. The discovery of modifications of these proteins allowsrecognition of previously unknown epitopes that can be used as the basisfor the selection of antibodies against the sulfonated regions of theseproteins for diagnostic or therapeutic purposes.

Thus, in one particular embodiment of the present invention, antibodiesare isolated or produced that recognize the sulfonated portions ofSclerostin. The antibodies may be specific for epitopes where the aminoacid is sulfonated. Alternatively, the antibody may recognize and bindonly to epitopes where the amino acid remains unsulfonated.

In another embodiment of the present invention, antibodies are isolatedor produced that recognize the sulfonation region of Sclerostin,regardless of whether the amino acid in this target region is sulfonatedor not. The antibodies of the present invention may be used alone or inconjunction with other previously described antibodies to Sclerostinthat may be specific to regions that do not comprise sulfonationmodification sites.

Another embodiment of the present invention is directed towardsdiagnostic utility, where the potential dependency of biologicalproperties on the presence or absence of sulfonation in these siteswould allow evaluation of clinical properties that may depend upon thedegree of sulfonation of these proteins. This may be used for theidentification of a disease condition or used to monitor the effects ofa therapeutic program.

In another embodiment of the present invention, antibodies against thesulfonate modified regions of Sclerostin may be administered to block orinterfere with binding of Sclerostin with an interacting protein,thereby reducing the functional activity of Sclerostin in a subject andproviding beneficial effects.

Furthermore, in another embodiment of the present invention, thediscovery of a linkage between the sulfonation state of Sclerostin andbiological activity presents an opportunity to use the sulfonatedregions as targets for intervention, where pharmaceutical compounds maybe used to affect the degree of sulfonation and thereby modulate thebiological activity of Sclerostin.

In another aspect of the present invention, potential differentialeffects of therapeutic compounds on sulfonated or unsulfonated forms ofSclerostin may now be addressed due to the discovery of sulfonationmodifications in Sclerostin. Consequently, virtual screening processesfor identification of compounds that bind to selected sites onSclerostin may now be carried out where the virtual model of the targetmolecule includes a substitution of the sulfonated amino acid for theunmodified form in Sclerostin, thereby altering projected bindingaffinities when compounds are screened.

Another embodiment of the present invention makes use of the fact thatsulfonation modifications are usually involved in protein-proteininteractions, and as such, the identification of sulfonationmodification sites in Sclerostin serves as an indication that theseregions can be indentified as previously unknown sites that participatein interactions with other protein molecules. Therefore, in anotherembodiment of the present invention, the synthesis of peptides thatcomprise the sulfonation region may have utility as modulators, alteringthe ability of Sclerostin to interact with its binding partners. Incarrying out such a role, both sulfonated and unsulfonated forms of suchpeptides may provide utility.

Lastly, interactions between Sclerostin and a binding partner areusually described in the context of heterodimeric complexes such asSclerostin/LRP or Sclerostin/BMP receptor. It is a further considerationof the present invention, however, that the discovery of sulfonationmodifications may have ramifications with regard to homodimericcomplexes that may take place between two Sclerostin monomeric units.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-1A, 1-1B, 1-1C, 1-2A, 1-2B and 1-2C show the identification ofsulfonated Tyrosines in Sclerostin by mass spectrometry. FIGS. 1-1Athrough 1-2C disclose SEQ ID NOS: 9 and 7, respectively, in order ofappearance.

FIG. 2 are results of treatment of Sclerostin with TPST-1 and asubsequent comparison between treated and untreated Sclerostin withregard to binding to the LRP5 receptor.

FIG. 3 shows the differential effects produced by treated and untreatedSclerostin with regard to Wnt-induced Alkaline Phosphatase expression.

FIG. 4 is a comparison between epitopes defined by Sulfonation sites ofSclerostin and epitopes previously described in the literature. FIG. 4discloses all sequences as SEQ ID NO: 10.

DESCRIPTION OF THE INVENTION

The present invention describes the discovery that Sclerostin, a ligandof various LRP receptors, can be sulfonated in at least two differentsites, Tyr₄₃ and Tyr₂₁₃ (using the UniProtKB Accession No. Q9BQB4 ofunprocessed Sclerostin as reference points). A further discovery is thatan ex vivo sulfonation treatment of a preparation of recombinantSclerostin results in an increase in the affinity of the Sclerostin tothe LRP5/6 receptor as well as an increase in its ability to suppressWnt induced expression of Alkaline Phosphatase. Various means may beemployed to determine the presence of sulfonation modifications inproteins of interest. As described in Example 2 below, mass spectrometry(mass spec) analysis was carried out using Sclerostin that was expressedin cells capable of carrying out post-synthetic modifications such assulfonation. It should be noted that the standard conditions that areusually employed in mass spec studies leads to a rapid loss of sulfonategroups. As such, when detection of sulfonated targets is desired,avoidance of acidic conditions and lower energy inputs are required inorder to increase the sensitivity of detection of sulfonated Tyrosinesin specimens of interest (Drake and Hortin, 2010). This type of analysismay lead to the identification of the presence of sulfonated Tyrosinesand in many cases the exact position of the sulfonated amino acid. Acaveat to be considered is that the simultaneous presence of bothsulfonated and unsulfonated Tyrosines for a given fragment does not giveany estimate for their relative proportions prior to analysis since theprocess is still liable to losses of sulfonate moieties, therebygenerating some unsulfonated Tyrosines de novo. Distinguishing betweenpre-existing and converted unsulfonated Tyrosines is problematic and assuch, mass spec serves best as a qualitative tool for whethersulfonation occurs at all at a given site.

Prior to carrying out the mass spec analysis, some of the Sclerostin wasused in a reaction with TPST-1 (Example 1) such that if any Tyrosinemodification sites were present in the sample they could be convertedinto a modified from. As described in the mass spec analysis describedin Example 2, the presence of sulfonation modifications was found inboth the treated and untreated samples of Sclerostin, indicating thatthe recombinant Sclerostin being tested had undergone sulfonationmodifications prior to secretion from the cells used for recombinantexpression. As noted above, however, the mass spec analysis candetermine the presence of sulfonation modifications, but is unable toprovide information on whether there is complete or partial modificationon the sulfonation sites. A protein having the appropriate sulfonationsequence may be a candidate for post-synthetic modification as it passesthrough the Golgi apparatus prior to secretion outside of a cell, butrecombinant expression systems are essentially abnormal states and theremay be undersulfonation of sites that normally would be fully converted.

As such, treated and untreated Sclerostin has been used in biologicalassays to investigate whether there were any differences in theproperties of Sclerostin after an in vitro treatment. As described inExample 3 and as shown in FIGS. 2 and 3, the differences seen with thein vitro treatment are consistent with a conclusion that some Tyrosinesin the sulfonation sites of the recombinant Sclerostin passed throughwithout being modified prior to secretion from cells, thereby makingthem available for in vitro modification and consequently the convertedSclerostins displayed an increased affinity for their binding partners,i.e., the original sample contains partially sulfonated Sclerostin andthe treated sample has an increased level of sulfonation. This effectcould likely be seen more dramatically if conditions were used such thata comparison was made with starting material that was completely ormostly lacking in sulfonation modifications prior to an in vitroreaction. Ways that this could be accomplished are the use of yeast orbacterial expression systems, treatment of recombinant expression cellswith chlorate prior to harvesting the protein or expression in cellsthat have been mutated to eliminate TPST activity such as thosedescribed by Westmuckett et al., 2008, Gen. Compar. Endocrine156:145-153. With regard to the chlorate treatment, it has beenpreviously shown that such treatment can strongly reduce the degree ofsulfonation in cells (Baeuerle and Huttner 186 BBRC 141; 870-877; Hortinet al., 1988 BBRC 150; 342-348; Mintz et al., 1994 J Biol Chem 269;4845-4852) and a bacterial or yeast host would lack any sulfonationsince they intrinsically lack the sulfotranferases responsible forTyrosine sulfonation (Kehoe and Bertozii 2007 Chemistry & Biology 7;R57-R61).

A further method of investigation is the use of a software programcalled Sulfinator that can predict the presence of a sulfonation sitewith 98% accuracy from the amino acid sequence alone. When the sequencesfrom Sclerostin were analyzed with this program, it successfullyidentified the amino terminal modification of Sclerostin at Tyr₄₃detected by MS analysis but missed the carboxy terminal modification atTyr₂₁₃. The failure in predictability is likely due to the Tyrosine inSclerostin that is modified at the carboxy end of Sclerostin being theterminal amino acid itself; since the Sulfinator program uses theneighboring sequences surrounding a Tyrosine for evaluating itslikelihood of being sulfonated, by definition, a terminal Tyrosine ofSclerostin would intrinsically lack sequences on one side. It should bepointed out that although the presence of a site predicted to be asulfonation site is likely dependable, there are incidents wheresulfonation modifications were unrecognized by the Sulfinator programbut later identified in physical studies (Onnerfjord et al., 2004 JBC279; 26-33, Hoffhines et al., 2006 JBC 281; 37877-37887). Nonetheless,the recognition of the Tyr₄₃ modification by the Sulfinator program isan independent confirmation of the sulfonation of this particular aminoacid in Sclerostin.

It should be understood that although sulfonated Tyrosines have beenobserved in many secreted ligands and their receptors, their presence isnot necessarily required and it is inappropriate to make any predictionsabout their presence in the absence of any investigational analysis. Asnoted above, it has been estimated that ˜1% of the Tyrosines in cellularproteins are modified Tyrosines (Huttner 1984 Methods Enzymol 107;200-223) which in turn has the implication that ˜99% of them would nothave this modification.

The discovery that Sclerostin, which participates in the Wnt signalingsystem, has modified amino acids offers unique methods of analysis aswell as therapeutic means. As described in earlier related patentapplications (2005/0196349; 2006/0030523; 2008/0119402, herebyincorporated by reference), compounds that block the interaction betweenLRP5/6 receptors and the Dkk and Sclerostin ligands can offer a varietyof useful therapeutic means. Since it has now been discovered that theamino acid sequence of Sclerostin can also comprise a post-syntheticsulfonation modification, compounds that have been previously tested foreffects on Sclerostin with regard to Wnt signaling may be retested usingseparate reaction mixtures or binding assays where either the modifiedor unmodified versions of these proteins are tested separately. As hasbeen pointed out earlier, some proteins exist as a mixture of sulfonatedand unsulfonated forms and previous experiments may have been based uponsuch a mixture, without recognition that the net effects might be acomposite of the individual effects on modified and unmodifiedSclerostin. Control over the particular form (sulfonated orunsulfonated) of Sclerostin will now allow investigation into whethercompounds are more or less effective with regard to using sulfonated orunsulfonated versions of Sclerostin in assays. The lack of recogntion ofthe potential presence of a mixture of different forms also allows forthe possibility that some effective compounds may have been missed dueto the use of Sclerostin preparations that had a preponderance of oneform over another.

Furthermore, the presence of a site that is involved in protein-proteininteractions is in itself a potential therapeutic target. Thus, a seriesof compounds can be surveyed to see if they specifically interrupt invitro or in vivo sulfonate modification of the Tyrosines in Sclerostin.Such pharmaceutical agents would have the potential for modifying thelevel of activity induced by Sclerostin by controlling the degree ofsulfonation and thereby their affinity in protein-protein interactions.Pharmaceutical reagents that may be used to disrupt sulfonationprocesses can include but not be limited to small molecules, peptides,organic molecules, cyclic molecules, heterocyclic molecules, lipids,charged lipids, glycolipids, polar lipids, non-polar lipids andaptamers. The compounds may be ones that have been designed to bind tothe surface of Sclerostin through a virtual screening process asdescribed in US Patent Application No. 2005/0196349. Contrariwise,compounds may be tested independently from virtual screening and testedstrictly on a random basis or they may be selected to have a physicalresemblance to compounds that result from virtual screening processes.In this process, a revised virtual structure of sclerostin may bedevised to takes into consideration the presence of the sulfonation ofamino acids. Such a process can also include the use of mutationalsubstitutions at the modification sites (see, for instance, Wu et al.,2005/0196349). Thus, a series of (selected or random) compounds may beassayed for an ability to eliminate or reduce sulfonation of Sclerostin,by any means that have previously been described for analysis and/ordetection of sulfonation of proteins. As a control, one or more proteinsthat also have sulfonation sites may be included to insure that theblockage of sulfonation is specific for the target of interest. Anymeans that have been described in the past for detection of the presenceof sulfonated Tyrosines may be used in this aspect of the presentinvention, thus for example, these means may be as complex as carryingout MS analysis to simpler methods such as incorporation of ³⁵S PAPS byTPST, immunoassays that use antibodies that recognize proteins withsulfonated Tyrosines irrespective of their context (Kehoe et al., 2006and Hoffhines et al., 2006), or antibodies that are specific for thesulfonated or unsulfonated forms of Sclerostin (as will be discussed inmore detail below). If desired, truncated versions of Sclerostin thatcomprise the target area of interest may also be used as substrates inassays as long as their biological structures/functions are similar tothe parent protein. In addition, peptides that may represent thesulfonation site of Sclerostin may also be used for evaluation.

Investigations into compounds that might interrupt sulfonation ofproteins has been previously described by Hortin et al., 1988 BBRC 150;342-348 where compounds were found that were non-specific in that theyinhibited sulfonation of proteins, oligosaccharides and proteoglycans(although with varying efficiencies). A similar study has been done morerecently by Kehoe et al., 2002 (Bioorg Med Chem Letters 12; 129-132)where two compounds were identified that inhibited sulfonation byTPST-2. Similar to the results published earlier by Hortin et al.,further testing showed that the inhibitors affected othersulfotransferases as well. Even if these inhibitors only affected theTPST reaction itself, however, this approach would indiscriminatelyblock sulfonation of a wide variety of different protein targets andthereby lead to potentially toxic effects. It should be noted thatknockout mice lacking either TPST-1 (Ouyang et al., 2002 JBC 277;23,731-23,787) or TPST-2 (Borghei et al., JBC 281; 9423-9431) activityare essentially viable but exhibit a variety of pleiotropic defects.Partial overlap in the functionality of the two TPST enzymes can be seenby experiments with a double knockout that is missing both TPST-1 andTPST-2 where most progeny died soon after birth and any survivors failedto thrive (Westmuckett et al., 2008). These double knockouts exemplify asituation that may be more akin to the presence of a general TPSTinhibitor. In addition, as noted previously, there are many proteinsinvolved in protein-protein interactions where sulfonation is obligatoryfor biological activity and some are involved in inflammatory responsesthat require sulfonation for functionality; as such, it may be that thedouble knockouts are phenotypically silent except under certainconditions where such responses would be induced or required. Targetingthe modification of a particular sulfonation target as described in thepresent invention is likely to be superior to efforts to blocksulfonation in general since it is likely to have more specific effectsthan a general blockage that may produce deleterious as well asbeneficial effects.

The identification of peptide sequences comprising a modified Tyrosinealso allows the use and design of artificial peptides that contain thesemodifications. Presumably these should have higher binding affinitiesthan their unmodified counterparts. Binding of these peptides to theprotein that normally interacts with the complete protein may produce avariety of effects. For instance, some of these peptides could act in apositive fashion by invoking the same response that the intact proteininduces. Contrariwise, the peptide could act as a competitive inhibitorand prevent the intact protein from binding. For example, a peptide withsequences from either the carboxy or amino end could reduce the abilityof Sclerostin to bind to a LRP receptor. Although the sulfonated peptidewould be the basis for the design, it is understood that the actualcomponents can be artificial equivalent of these peptides. Examples ofcompounds made with such components can comprise but not be limited tothe peptide mimetics described in related pending U.S. patentapplication Ser. No. 11/097,518, as well the substitution of dextroisomers instead of the normal levo forms and peptidomimetics such asthose described in Hammond et al., 2006 Chem & Biol 13; 1247-1251. Otherexamples of analogs that may find use with the present invention are“unnatural amino acids” where in it is understood that in the context ofthe present invention “unnatural amino acids” refers to amino acids thatare not genetically encoded i.e. they are not represented by anucleotide triple codon. This would include the dextro isomers discussedabove as well as other amino acids such as Aib (amino-isobutyric acid),bAib (3-aminoisobutyric acid), Nva (norvaline), 13-Ala, Aad(2-amino-adipic acid), bAad (3-am inoadipic acid), Abu (2-am inobutyricacid), Gaba (γ-aminobutyric acid), Acp (6-aminocaproic acid), Dbu(2,4-diaminobutyric acid), TMSA (trimethylsilyl-Ala), aIle(allo-Isoleucine), Nle (Norleucine), tert.Leu, Cit (Citrulline), Orn,Dpm (2,2′-diaminopimelic acid), Dpr (2,3-diaminopropionic acid), α- orβ-Nal, Cha (cyclohexyl-Ala), hydroxy-proline, Sar (Sarcosine) etc.,cyclic amino acid units and N^(α)-alkylated amino acid units, e.g. MeGly(N^(α)-Methyl-glycine), EtGly (N^(α)-ethylglycine) and EtAsn(N^(α)-ethyl-asparagine). Accordingly, synthetic peptides can be madethat include one or more of these unnatural amino acids.

Another series of reagents that may be effective are antibodies directedto the sulfonation site. In the first place, the identification of thesulfonation site offers evidence that the site is likely to be involvedin protein-protein interactions. Thus, for instance, the particularportion of the Sclerostin protein involved in interaction with LRP5/6has not been clearly identified, but the discovery of the sulfonationsite of Sclerostin in the amino terminal sequences described in Example2 provides a novel target for antibody binding that might affect theinteraction of Sclerostin with LRP5/6 that is different from theSclerostin sequences previously postulated by Ververka et al., 2009 JBC284; 10,890-10,900, Weidauer et al., 2009 BBRC 380; 160-165 and Krumlaufin U.S. Pat. No. 7,585,501. In one embodiment of the present invention,an epitopes is five amino acids or greater. In another embodiment, theepitope encompasses ten or more amino acids. A comparison of theidentity and location of potential epitopes of the present invention andsequences used as epitopes in prior art is given in FIG. 4. Theunderlined regions adjacent to Tyr₄₃ and Tyr₂₁₃ in FIG. 4 are onlyintended to render a visual aid in comparing the regions of the presentinvention with previously described art and not intended to delineatethe epitope itself. Antibodies may be generated that are specific foreither the sulfonated or unsulfonated form of Sclerostin and includeeither Tyr₄₃ or Tyr₂₁₃ as part of their epitope. Utility may also befound for antibodies that do not distinguish between the sulfonated andunsulfonated forms, but are still specific for the surrounding aminoacids at the Sclerostin sulfonation site. The foregoing antibodies mayfind use as therapeutic reagents that disrupt interaction betweenSclerostin and LRP5/6 as well as other binding partners of Sclerostin.In the case of antibodies that are specific for either sulfonated orunsulfonated forms, a finer degree of control can be exerted overphysiological processes, since each type of antibody will be directedtowards a subpopulation of the target protein. As such, an ability totarget only the sulfonated form will leave the activity of theunsulfonated from intact and vice versa for an antibody to theunsulfonated from. This is a level of discrimination that would not beproduced by antibodies described previously for Sclerostin. On the otherhand, an antibody of the present invention that is generic in the senseof being independent of the sulfonation state of Sclerostin, may alsohave therapeutic utility because the sites where modifications takeplace may have more significance than previously recognized, and thus,these regions are novel epitopes that are useful as targets forimmunotherapy.

Development and isolation of antibodies that are targeted to thesulfonation regions discovered and described in the present inventionmay be carried out by any of the means that have been describedpreviously, including those taught in Bundgaard et al., 2008; Hoffhineret al., 2006; Kehoe et al. 2006, Craig et al., 2009 Hybridoma 28;377-381; U.S. Pat. No. 7,585,501, US Patent Application 2004/0009535 andUS Patent Application 2009/02130113, all of which are incorporated byreference. One source of antigens that may be used for this purpose canbe artificial peptides that represent the sulfonated sequences; thesecan be obtained from a wide variety of commercial sources that providecustom made peptides. The peptide or peptides used for immunization maybe modified or unmodified, depending upon whether the antibody isdesired to recognize the modified or unmodified epitope. Post-syntheticmodifications can be carried out either chemically or by in vitromodification by TPST-1. Screenings of antibody libraries can then becarried out to determine the nature of the recognition such that it isspecific for the sulfonated version of the target protein, theunsulfonated form or is independent of the state of sulfonation. Inaddition to such custom libraries, pre-existing libraries such as theHuCal phage library is commercially available from AbD Serotec (Raleigh,N.C.) and is advertised as having more than 15 billion functional humanantibody specificities. Another commercially available library comprisescamelid derived antibodies and is available from Ablynx, Ghent, Belgium.These libraries have the advantage of not requiring any particularimmunogen prior to screening. Screenings of this library may also becarried out as discussed above. The antibody of the present inventionmay take any form that is described for use in immunodetection orimmunotherapy. For instance, the antibody may be polyclonal, monoclonal,chimeric, human, humanized, bispecific, multispecific, primatized or anantibody fragment. Antibody fragments that me be of use in the presentinvention may comprise but not be limited to is Fab, ScFv, Fab′,F(ab′)₂, Fv, Fv(ab)₂ or aggregates thereof.

The presence of a sulfonation group should in itself be sufficient todefine part of an epitope. In an analogous fashion for anotherpost-synthetic modification, the literature is replete with a largenumber of antibodies that are dependent on targets being either inphosphorylated or unphosphorylated forms and these form the basis ofnumerous assays for kinase activity. Furthermore, as describedpreviously, the presence or absence of such small chemical moieties as aphosphate or sulfonate group can have profound effects upon activity,thus validating the ability of biological partners to be able torecognize the differences between modified and unmodified forms.Specific examples of the search and identification of antibodies thatare specific to epitopes of target proteins comprising a sulfonatedTyrosine have been described by Bundgaard et al., cited above. In afurther example, an antibody (Mab15) that was selected for recognizingthyrotrophin receptor (TSHr) was found to have an epitope that was onlyfound in mature forms of its target protein suggesting that some form ofprocessing was required to create the appropriate epitope (Costagliolaet al., 2002 EMBO J 21; 504-513). In vivo treatment of cells withchlorate (which as mentioned earlier reduces sulfonation modifications)resulted in production of a mature protein that was now unrecognizableby Mab15 indicating that the antibody was able to distinguish betweenthe sulfonated and unsulfonated forms of its epitope and would only bindto the sulfonated version. Thus, even though it was not originallyselected for this feature, the use of sulfonated antigens allowedisolation and identification of an antibody specific for a sulfonateepitope in this target.

Although peptides may be used for the generation of linear epitopes,antibodies can also be found that recognize a three-dimensional set ofdeterminants (sometimes referred to as interrupted epitopes ornon-linear epitopes) and development and isolation of these types ofantibodies can be carried out by using three-dimensional antigens suchas the entire protein of interest or selected fragments as immunogens.Such antibodies may also be realized from screening of pre-formedlibraries that are independent of an immunogen. Screening can then becarried out for an ability to distinguish between sulfonated andunsulfonated versions of the protein of interest. For a discussion onthe use of conformationally derived epitopes, see Van Regenmortel 1998,J Immunol Methods 216; 37-48, Villen et al., 2001 Biologicals 29;265-269, Moreau et al., 2006 Bioinformatics 22; 1088-1095 and Huang andHonda 2006 BMC Immunology 7; 7.

In addition to acting as pharmacological agents, the development ofantibodies directed against the sulfonation site of Sclerostin, may alsofind use for analytical or diagnostic purposes for evaluating thepresence of sulfonated proteins and/or the extent of sulfonation. Asdescribed previously, shifts in the level of sulfonation levels haspreviously noted to be a feature of gastrin and cholcystokinin in cancercells (Rehnfeld, 1990). The protein samples may be products that areexcreted in the media or they may be derived from cell extracts. Bythese means, evaluation of physiological levels of sulfonation ofSclerostin can be carried out with biological specimens. These may beused in a variety of ways to compare specimens that differ from eachother in terms of origin, treatment or physiological conditions. Anantibody specific for a sulfonated form of a target protein may be usedalone for this purpose or it may be combined in an assay that furtherincludes an antibody directed towards the unsulfonated form or anantibody that recognizes both sulfonated and unsulfonated forms. Inreference to the latter, an ability to recognize both sulfonated andunsulfonated forms may be a property of an antibody that recognizes theepitope where the sulfonation is located but is generically independentof the sulfonation state, or it can an antibody that lacks relevance tothe sulfonation state by recognizing an epitope that is located outsideof the modification region of Sclerostin.

As discussed above, antibodies of this nature may also be used toevaluate in vitro assays of sulfonation where they may be used tomonitor conversion of the unsulfonated form into the modified form.These antibodies may also be used alone or in conjunction withantibodies that recognize an epitope specific for the unsulfonated formand/or for antibodies to an epitope in an amino acid sequence differentfrom the sulfonation sequence. Thus, for instance, an antibody that isspecific for the sulfonated form of Sclerostin may be used inconjunction with an antibody that is specific for an unsulfonated regionof Sclerostin for normalization purposes. In another example of use, anantibody that is specific for the unsulfonated form can be used inconjunction with an antibody that recognizes the same region butessentially offers no discrimination between the sulfonated andunsulfonated forms of the antigen. Alternatively, two antibodies can beused where one is specific for the sulfonated form and another is forthe unsulfonated form.

Furthermore, although the binding of Sclerostin to an LRP receptor isresponsible for biological effects, it is also known that Sclerostininteracts with other proteins such as BMPs (Bone Morphogenic Proteins)(Winkler et al., 2003 EMBO J 22; 6267-6276), Noggin (Winkler et al.,2004 J Biol Chem 279; 36293-36298) and “Cysteine-rich protein 61” (Craiget al 2010 (BBRC 392; 36-40). As such, the discovery of the sulfonatedamino acids in Sclerostin allows application of the present invention tointeractions between Sclerostin and these other proteins as well as theinteractions with LRP receptors. Additionally, although the Tyrosinemodifications have been discussed in terms of alterations of aSclerostin's affinity for a binding partner in a heterodimericinteraction, dimerization is also an example of a protein/proteininteraction and as such, a homodimeric Sclerostin interaction may alsobe influenced by sulfonation modifications, and Sclerostin itself,should be included in the potential list of binding partners forSclerostin. The degree of dimerization may have further effects withregard to binding to other proteins, where the affinity of a dimericprotein may be higher than that of a monomeric form. For instance, seeJekel et al., Biochimica Biophica Acta 1996 1291; 195-198 where theaffinity of a dimerized antigenic peptide is higher than the monomericform with regard to binding to an antibody. In another instance, TNF-αexists in trimeric form and binds to three receptors simultaneously(Banner et al., 1993, Cell, 73:431-445). Since dimerization ormultimerization of proteins may be affected by sulfonation processes,the methods above may also be applied to homodimeric interactions whenthe ability of a compound to affect sulfonation is being analyzed.Assays that measure the ability of sulfonated and unsulfonatedSclerostin to form a complex with a binding partner may also be carriedout with another Sclerostin molecule as the intended binding partner.Antibodies may also be developed that are specific to dimers as comparedto monomers as previously described by Raven et al., in US Patentapplication No. 20050163776. Selectivity may be carried out by testingfor the ability to react with dimers and then counter-selecting byeliminating antibodies that exhibit cross-reactivity with the monomericform.

Antibodies directed against sulfonation sites in Sclerostin andpharmaceutical agents that disrupt or inhibit sulfonation of Sclerostinmay find therapeutic utility with a variety of “Sclerostin-relateddisorders”. The compositions and methods of the present invention areparticularly suitable for treating, preventing or diagnosingSclerostin-related disorders and/or aberrant bone mineral densitydisorders, e.g., osteoporosis. Compositions of the present invention mayalso be useful for improving outcomes in orthopedic procedures, dentalprocedures, implant surgery, joint replacement, bone grafting, bonecosmetic surgery and bone repair such as fracture healing, nonunionhealing, delayed union healing and facial reconstruction. One or morecompositions may be administered before, during and/or after theprocedure, replacement, graft, surgery or repair.

As used herein, “a Sclerostin-related disorder” includes disorders inwhich bone mineral density (BMD) is abnormally and/or pathologically lowrelative to healthy subjects. Disorders characterized by low BMD and/orbone fragility include but are not limited to primary and secondaryosteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (01),avascular necrosis (osteonecrosis), fractures and implant healing(dental implants and hip implants), bone loss due to other disorders(e.g., associated with HIV infection, cancers, or arthritis). Other“Sclerostin-related disorders” include but are not limited to rheumatoidarthritis, osteoarthritis, arthritis, and the formation and/or presenceof osteolytic lesions.

As used herein, “a Sclerostin-related disorder” includes conditionsassociated with or characterized by aberrant Sclerostin levels. Theseinclude cancers and osteoporotic conditions (e.g., osteoporosis orosteopenia), some of which overlap with “Sclerostin-related disorders”as defined herein. Sclerostin-related cancers can include myeloma (e.g.,multiple myeloma with osteolytic lesions), breast cancer, colon cancer,melanoma, hepatocellular cancer, epithelial cancer, esophageal cancer,brain cancer, lung cancer, prostate cancer, or pancreatic cancer, aswell as any metastases thereof.

A “Sclerostin-related disorder” can also include renal andcardiovascular conditions, due at least to Sclerostin's expression inthe kidney and cardiovasculature. Said disorders include but are notlimited to such renal disorders as glomerular diseases (e.g., acute andchronic glomerulonephritis, rapidly progressive glomerulonephritis,nephrotic syndrome, focal proliferative glomerulonephritis, glomerularlesions associated with systemic disease, such as systemic lupuserythematosus, Goodpasture's syndrome, multiple myeloma, diabetes,polycystic kidney disease, neoplasia, sickle cell disease, and chronicinflammatory diseases), tubular diseases (e.g., acute tubular necrosisand acute renal failure, polycystic renal diseasemedullary spongekidney, medullary cystic disease, nephrogenic diabetes, and renaltubular acidosis), tubulointerstitial diseases (e.g., pyelonephritis,drug and toxin induced tubulointerstitial nephritis, hypercalcemicnephropathy, and hypokalemic nephropathy) acute and rapidly progressiverenal failure, chronic renal failure, nephrolithiasis, gout, vasculardiseases (e.g., hypertension and nephrosclerosis, microangiopathichemolytic anemia, atheroembolic renal disease, diffuse corticalnecrosis, and renal infarcts), or tumors (e.g., renal cell carcinoma andnephroblastoma).

Said disorders also include but are not limited to such cardiovasculardisorders as ischemic heart disease (e.g., angina pectoris, myocardialinfarction, and chronic ischemic heart disease), hypertensive heartdisease, pulmonary heart disease, valvular heart disease (e.g.,rheumatic fever and rheumatic heart disease, endocarditis, mitral valveprolapse, and aortic valve stenosis), congenital heart disease (e.g.,valvular and vascular obstructive lesions, atrial or ventricular septaldefect, and patent ductus arteriosus), or myocardial disease (e.g.,myocarditis, congestive cardiomyopathy, and hypertrophic cariomyopathy).

According to a further embodiment of the invention, the antibodies andother pharmacological agents of the present invention may be employed asadjunct or adjuvant to other therapy, e.g. a therapy using a boneresorption inhibitor, for example as in osteoporosis therapy, inparticular a therapy employing calcium, a calcitonin or an analogue orderivative thereof, e.g. salmon, eel or human calcitonin, calcilytics,calcimimetics (e.g., cinacalcet), a steroid hormone, e.g. an estrogen, apartial estrogen agonist or estrogen-gestagen combination, a SERM(Selective Estrogen Receptor Modulator) e.g. raloxifene, lasofoxifene,bazedoxifene, arzoxifene, FC1271, Tibolone (Livial®), a SARM (SelectiveAndrogen Receptor Modulator), a RANKL antibody (such as denosumab), acathepsin K inhibitor, vitamin D or an analogue thereof or PTH, a PTHfragment or a PTH derivative e.g. PTH (1-84) (such as Preos™), PTH(1-34) (such as Forteo™), PTH (1-36), PTH (1-38), PTH (1-31)NH2 or PTS893. According to another embodiment, the antibodies of the inventionmay be employed in combination with other current osteoporosis therapyapproaches, including bisphosphonates (e.g., Fosamax™ (alendronate),Actonel™ (risedronate sodium), Boniva™ (ibandronic acid), Zometa™(zoledronic acid), Aclasta™/Reclast™ (zoledronic acid), olpadronate,neridronate, skelid, bonefos), statins, anabolic steroids, lanthanum andstrontium salts, and sodium fluoride. When pharmacological agents ofantibodies of the present invention are administered together withanother agent, the two can be administered in either order (i.e.sequentially) or simultaneously.

The examples which follow are set forth to illustrate various aspects ofthe present invention but are not intended in any way to limit its scopeas more particularly set forth and defined in the claims that followthereafter.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1 In Vitro Sulfonationof Sclerostin

Human Sclerostin (25 μg R&D Systems, Minneapolis, Minn.)) wasreconstituted in 100 μl of 100 mM MES pH=7.0. Sulfonation was carriedout by mixing 50 μl (12.5 μg) human Sclerostin and 22.5 μl (10.0 μg) ofhuman TPST1 (R&D Systems) with 125 μL of assay mix [78.87 mM MES pH=7.0,2.5 mM MgCl₂, 2.5 mM MnCl₂ 1.25 mM CaCl₂ and 200 μM PAPS (Sigma)].Incubation was carried out for 1.5 hrs at 37° C. Buffer was thenexchanged into 10 mM Tris pH=7.5 using protein desalting spin columns(Pierce Biochemicals, Rockford, Ill.).

Example 2 Detection of Sulfonation Modifications by MS Analysis

Peptides from the Sclerostin from Example 1 as well as untreatedSclerostin was digested with either trypsin or GluC and loaded onto aC18 column followed by injection into a LTQ mass spectrometer. In thefirst analysis, the mass spectrometer was instructed to make MS/MS ofall eluting peptides. The resulting data was analyzed and three peptidesfrom Sclerostin containing tyrosines were identified: LGEYPEPPPELE (SEQID NO: 1), YVTDGPCR (SEQ ID NO: 2) and ANQAELENAY (SEQ ID NO: 3). In thesecond analysis, targeted analysis was performed where the massspectrometer was instructed to only do MS/MS on masses corresponding tothe putative sulfonated tyrosine containing peptides. For the GluCsample, the mass spectrometer was set to perform MS/MS at m/z 725.6, themass of the doubly charged peptide LGEYPEPPPELE (SEQ ID NO: 1) plussulfation, at a normalized collision energy for CID at 2%, 4% or 10% andan MS3 of the highest fragment in each of the three MS/MS. In thetryptic sample, MS/MS was performed at m/z 602.0, the mass of the doublycharged peptide ANQAELENAY (SEQ ID NO: 3) plus sulfonation, and at m/z524.5, the mass of the doubly charged peptide YVTDGPCR (SEQ ID NO: 2)plus sulfonation, at a normalized collusion energy for CID at 2%, 4% and10% and an MS3 of the highest fragment in each of the three MS/MS. Bothpeptides at m/z 725.6 and 602.0, corresponding to sulfonated peptideLGEYPEPPPELE (SEQ ID NO: 1) and ANQAELENAY (SEQ ID NO: 3), respectively,showed a neutral loss of 80 Da (40 Da for a 2+ ion) at 10% CE whichsuggests that these peptides were sulfonated, whereas at 2% and 4%, theloss was not very pronounced (see FIG. 1). The neutral loss fragmentswere subsequently fragmented and produced the expected MS/MS for theexpected peptides. The MS/MS at m/z 524.5 did not show such a loss (datanot shown). Essentially the same results were seen for both theuntreated sclersotin and the Sclerostin from Example 1 indicating thepresence of sulfonation modifications in Sclerostin prior to the invitro reaction with TPST-1 in Example 1. Furthermore, although aphosphate addition at this site would also result in a shift of ˜80 kdhigher weight, further tests showed that the modifications at thesesites exhibited the chemical lability typical of a sulfonationmodification.

Example 3 Biological Effects of In Vitro Sulfonation of Sclerostin A)Effects of Sulfonation on Binding of Sclerostin to LRP5 1) Preparationof AlkPhos Labeled LRP5

293T cells were seeded into 9 cm dishes. The next day, each dish wastransfected with 12 μg of LRP5R1/2AP construct using Lipofactamine Plus(Invitrogen, Carlsbad, Calif.) according to the manufacturer'sinstructions. LRP5R1/2-AP is a nucleic acid construct that expressesLRP5 extracellular domains 1 and 2 fused to alkaline phosphatase. 48hours after transfection, the supernatant of the culture was collectedas LRP5R1/2AP conditioned medium and concentrated 20 times using aCentricon unit (Millipore, Billerica, Mass.) and stored at −80° C.

2) Binding of AlkPhos-LRP5 to Sclerostin

Various amounts of unmodified Sclerostin or the in vitro treatedSclerostin from Example 1 were diluted into 80 μl of TBST buffer andadded to individual wells of 96 well plates. After overnight incubation,unbound proteins were removed after which point the coated plates wereblocked with 3% nonfat milk in PBS. The plates were than decanted and0.5×LRP5R1/2AP conditioned medium was added to the plates. After 2.5hours, the conditioned medium was removed and the 96 well plates werewashed five times for three minutes with TBST. The Alkaline Phosphataseactivity in each well was then determined using the Tropix luminescenceassay kit (Invitrogen, Carlsbad, Calif.).

3) Results of the Binding Assay

As seen in FIG. 2, the Sclerostin treated in vitro with TPST showed amarked increase in the amount of AlkPhos-LRP5 bound to the plates whencompared to the untreated Sclerostin. These results are best interpretedas evidence that there is an increase in the binding affinity of thetreated protein compared to the starting material. These comparativeresults were repeated with the mouse versions of Sclerostin (not shown)and showed essentially similar results although the basal levels of theproteins were different for each source.

B) Effects of Sulfonation on the Ability of Sclerostin to Block WntInduced Expression of Alkaline Phosphatase 1) Induction of AlkalinePhophatase Activity

Growing cultures of 10T1/2 cells were washed with PBS and trypsinizedfor 5 minutes. Cells were resuspended at a concentration of 6×10⁵cells/ml and 10 μl were seeded into individual wells of a Costar 96 wellplate (Corning, Inc.). Wnt 3a and either the untreated Sclerostin or theSclerostin from Example 1 were added and the plates were incubated at37° C. for 24 hours. 50 μl of universal lysis buffer (from theLuciferase Reporter Gene Assay, Roche Applied Science, Indianapolis,Ind.) was added to each well at ambient temperature for 5 minutes.Detection of alkaline phosphatase was measured by the addition of 50 μlof ready-to-use CPSD with Sapphire Enhancer (Applied Biosystems)followed by an incubation at ambient temperature for 25 minutes.

2) Results of the Assay

As seen in FIG. 3, the Sclerostin sulfonated in Example 1 gave similarresults compared to the untreated Sclerostin except at the highest levelof Sclerostin input where there was a significantly (P=0.006) moreefficient blockage of Wnt induced alk phos activity. This result for thehighest level of Sclerostin may be a result of the increase in thebinding affinity of the treated protein compared to the startingmaterial as seen in FIG. 2.

It should be noted that the Sclerostin used in these experiments wasderived from recombinant clones in mammalian cell lines. Consequently,as seen in the MS results in Example 2, there is a significantpopulation of Sclerostin proteins that already have pre-existingsulfonation modifications. Thus, the positive effects seen in theexperiments above is the result of conversion of any remainingunsulfonated forms into the sulfonated version by TSPT-1.

Example 4 Evaluation of Sclerostin Sequences with “Sulfinator” Program

The “Sulfinator” program is an online methodology of predicting thepresence of sites in proteins that are substrates for tyrosinesulfonation (Monigatti et al. 2002 Bioinformatics 18; 769-770). Whenthis program was applied to the human Sclerostin sequence (UniProtKBAccession No. Q9BQB4), the amino acid sequence ELGEYPEPPPELENNK (SEQ IDNO: 4) in the N terminal region of Sclerostin was identified ascorresponding to a Tyrosine sulfonation site with sulfonation takingplace with Tyros in agreement with the MS results from Example 2. Thecorresponding sequences in the mouse and rat are GLGEYPEPPPENNQTM (SEQID NO: 5) and GLREYPEPPQELENNQ (SEQ ID NO: 6), respectively (UniProtKBAccession No Q99P68 and Q99P67) where differences in the amino sequenceare underlined. Evaluation of the mouse and rat Sclerostin sequences bythe Sulfinator program revealed that the rat protein should also besulfonated (and at the corresponding Tyr residue) while the mousesequence did not show a positive result. It should be noted, however,that part of the criteria used by the Sulfinator program is contextualneighboring amino acid sequences and when the oligopeptideGLGEYPEPPPENNQTM (SEQ ID NO: 5) from the mouse Sclerostin sequences wasindependently tested, it was indicated as being potential site forsulfonation. The loose structure at the amino terminal end of Sclerostin(to be discussed below) is likely responsible for the oligopeptideSulfinator results of mouse Sclerostin being in agreement with thebinding assay results.

The region of Sclerostin involved in binding to LRP5/6 is not preciselyknown. It has been described as “Finger 2” (˜aa's 115-147) by Weidaueret al., (2009 BBRC 380; 160-165) and “Loop 2” (˜aa's 86-112) by Veverkaet al., (2009 JBC 284; 10,890-10,900) where amino assignments are basedon the mature protein. It can be seen that neither putative locationcorresponds to the Tyr₄₃ site. Nonetheless, a visualization of thepredicted 3-dimensional structure shows that Tyr₄₃ is part of a looselyorganized peptide strand that could located in proximity with thebinding site in “loop 2” predicted by Ververka et al. As such, it ispossible that the amino terminal portion of Sclerostin also participatesin binding of Sclerostin to LRP5/6 and sulfonation may have effects onthis particular protein/protein interaction. Further support is fromU.S. Pat. No. 7,585,501 where the Tyr₄₃ site is a short distance awayfrom an additional Sclerostin sequence (#15) that was described asparticipating in binding with LRP5/6. This point is illustrated furtherin FIG. 4.

Example 5 Peptides Derived from Sulfonation Sites

Peptides from the sulfonation modification sites regions may be usefulin modulating protein-protein interactions between a sulfonated proteinand a binding partner. Thus, for example, the sequences ELGEYPEPPPELENNK(SEQ ID NO: 4) and KANQAELENAY (SEQ ID NO: 7) from Sclerostin can beused to artificially synthesize peptides that can be used as therapeuticcompounds. Both modified and unmodified versions of these peptides canbe made and tested to see which ones are more effective and if they areequivalent in potency.

Example 6 Development of Antibodies Specific for Sulfonated Proteins

Antibodies that are specific for Sclerostin can be developed usingpeptides derived from the recognition sequences described in Examples 2and 5. In FIG. 4, the sites previously described for use as epitopes forSclerostin antibodies is compared with the sulfonation sites describedin Example 2. Unmodified peptides can be designed and obtained fromnumerous commercial sources. Post-synthetic modifications can then becarried out either chemically or by in vitro modification by TPST-1.These antigens can then be used to obtain antibodies using methodstaught in Bundgaard et al., 2008; Hoffhiner et al., 2006 Kehoe et al.2006, U.S. Pat. No. 7,585,501, US Patent application 2004/0009535 and USPatent Application 2009/02130113. Screenings can be carried out todetermine the nature of the recognition such that it is specific forsulfonation of only the target protein. A similar program can be carriedout with analogous peptides that remain unmodified; these can be used toobtain antibodies that are specific for the unmodified version of thetargets. Screenings can also be based upon an ability to bind to thespecific region of the Sclerostin sulfonation, but the affinity of theprotein is for both sulfonated and unsulfonated versions of the antigentarget.

The discovery of a sequence in Sclerostin that comprises a sulfonatemodified Tyrosine provides information concerning previously unknownepitopes in Sclerostin that may be used to generate novel antibodiesthat target these sites. For this purpose, a peptide can be used thatcomprises the sequence ELGEYPEPPPELE (SEQ ID NO: 8) where the Tyrosineis modified to comprise a sulfonate group in order to generate anantibody that targets the sulfonated Tyrosine site at the amino end ofSclerostin. This modification can be carried out either chemically or bytreatment with TPST-1 and PAPS. Another peptide, comprising the sequenceKANQAELENAY (SEQ ID NO: 7) (where the Tyrosine is also modified bysulfonation) can be used to generate an antibody to the sulfonatedTyrosine site at the carboxyl end of Sclerostin. Generation andisolation of an antibody can then be carried out by the methodsdescribed by Bundgaard et al., 2008 in conjunction with the methodstaught in U.S. Pat. No. 7,585,501, US Patent Application 20040009535 andUS Patent Application 20090130113, all of which are incorporated byreference.

When using a peptide with a sulfonated Tyrosine as the immunogen,resultant antibodies can display a variety of different affinities. Forexample, in an article giving the protocol for generating antibodiesagainst peptides containing a phosphorylated Tyrosine, the point ismade: “Such an immunization will generate an immune response with atleast four components: (1) anti-carrier protein reactivity, (2) generalantiphosphotyrosine reactivity, (3) phosphorylation-independentanti-peptide reactivity and (4) phosphorylation-dependent anti-peptidereactivity.” (DiGiovanna et al., 2002 Current Protocols in Cell Biology16.6.1-16.6.18). As such, this article points out that even when using apeptide with the appropriate modification, antibodies can be generatedthat may only require the appropriate amino acid sequence and ignore thepresence or absence of a modified Tyrosine. Consequently, many of thepast efforts to isolate an antibody against a phoshphorylated peptidehave included a counter-selection step to eliminate antibodies that bindto the unphosphorylated version of the target peptide/protein.

In contrast, although it is a goal of the present invention to generateand isolate antibodies that are specific for a protein that has asulfonated tyrosine, utility is also found during such a search toidentify and isolate antibodies that are specific for the sulfonatedTyrosine site but that are also independent of the sulfonation state ofthe target protein. Thus in parallel, identification processes can becarried out that initially are identified in terms of an the ability tobind to the region encompassed by the sulfonation modifications and thena secondary screening can be carried out for a) antibodies that have theability to detect only epitopes that include the sulfonationmodification and b) antibodies that are independent of the sulfonationstatus of the target region.

Many obvious variations will no doubt be suggested to those of ordinaryskill in the art in light of the above detailed description and examplesof the present invention. All such variations are fully embraced by thescope and spirit of the invention as more particularly defined in theclaims that now follow.

1-130. (canceled)
 131. A method for production of a monoclonal antibodyspecific for a sulfonation site in Sclerostin, comprising the steps of:immunizing an animal with a synthetic peptide derived from theSclerostin amino acid sequence, wherein said peptide comprises asulfonation site in said Sclerostin that comprises a tyrosinecorresponding to Tyr43 or Tyr213 of human Sclerostin; isolating antibodyproducing cells from said immunized animal; fusing the isolated antibodyproducing cells with myeloma cells to form antibody producing hybridomacells; and screening the hybridoma cells to identify hybridoma cellsthat produce antibodies that discriminate between sulfonated andunsulfonated forms of said sulfonation site of Sclerostin.
 132. Themethod of claim 131, wherein said tyrosine is sulfonated in thesynthetic peptide.
 133. The method of claim 131, wherein said tyrosineis not sulfonated in the synthetic peptide.
 134. The method of claim131, wherein said tyrosine corresponds to Tyr43 of human Sclerostin.135. The method of claim 134, wherein said tyrosine is sulfonated in thesynthetic peptide.
 136. The method of claim 134, wherein said tyrosineis not sulfonated in the synthetic peptide.
 137. The method of claim131, wherein said tyrosine corresponds to Tyr213 of human Sclerostin.138. The method of claim 137, wherein said tyrosine is sulfonated in thesynthetic peptide.
 139. The method of claim 137, wherein said tyrosineis not sulfonated in the synthetic peptide.
 140. The method of claim131, wherein the synthetic peptide is selected from the group consistingof: (a) ELGEYPEPPPELE (SEQ ID NO:8), wherein tyrosine is sulfated; (b)ELGEYPEPPPELE (SEQ ID NO:8), Wherein tyrosine is not sulfated; (c)KANQAELENAY (SEQ ID NO:7), wherein tyrosine is sulfated; and (d)KANQAELENAY (SEQ ID NO:7), wherein tyrosine is not sulfated.
 141. Themethod of claim 140, wherein the peptide is ELGEYPEPPPELE (SEQ ID NO:8),wherein tyrosine is sulfated.
 142. The method of claim 140, wherein thepeptide is ELGEYPEPPPELE (SEQ ID NO:8), wherein tyrosine is notsulfated.
 143. The method of claim 140, wherein the peptide isKANQAELENAY (SEQ ID NO:7), wherein tyrosine is sulfated.
 144. The methodof claim 140, wherein the peptide is KANQAELENAY (SEQ ID NO:7), whereintyrosine is not sulfated.